Effect and mechanism of thymosin beta 4 on spinal cord-derived neural stem /progenitor cell injury induced by oxidative stress

Abstract

To investigate the role and mechanism of thymosin beta 4 (Tβ4) in oxidative stress injury of spinal cord-derived neural stem/progenitor cells (NSPCs) induced by hydrogen peroxide (H2O2). NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), H2O2 group (NSPCs cells damaged by 500 μM H2O2), Tβ4 -3 groups (NSPCs were treated with 1, 2.5, 5 μg/ml Tβ4 on the basis of H2O2 treatment) and TAK-242 group [NSPCs were treated with 5 μg/ml Tβ4 and Toll-like receptor 4(TLR4) inhibitor TAK-242 on the basis of H2O2 treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with H2O2 and Tβ4. The expression of Tβ4, TLR4, MyD88 were detected by qRT-PCR and Western blot. Cell viability was detected by MTT assay and lactate dehydrogenase(LDH) assay kit. Ca2+ concentration was detected by Fluo-3/AM probe method. The apoptosis of NSPCs was detected by flow cytometry and Caspase-3 and Caspase-9 kits;reactive oxygen species (ROS), superoxi dedismu-tase dismutase(SOD) activity and glutathione (GSH) content were detected by corresponding kits. Interleukin(IL)-6 and IL-1β were detected by enzyme-linked immunosorbent assay. The expression of Tβ4 was decreased in H2O2 injured NSPCs(P<0.05). Compared with H2O2 group, the cell viability and Ca2+ concentration was significantly increased, release of LDH and apoptosis were significantly decreased, production of ROS and pro-inflammatory cytokines were significantly decreased, and the expression levels of TLR4 and MyD88 protein were significantly decreased in Tβ4-3 groups and TAK-242 group (P<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (P<0.05), while after treatment with Tβ4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (P<0.05). Tβ4 attenuates H2O2-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways.